Evgenios Goussetis

Intracoronary Infusion of CD133+ and CD133−CD34+ Selected Autologous Bone Marrow Progenitor Cells in Patients with Chronic Ischemic Cardiomyopathy: Cell Isolation, Adherence to the Infarcted Area, and Body Distribution

Central issues in intracoronary infusion (ICI) of bone marrow (BM)‐cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM‐progenitors. We could immunomagnetically isolate 0.8 ± 0.32 × 107 CD133+ cells and 0.75 ± 0.24 × 107 CD133−CD34+ cells from 310 ± 40 ml BM. After labeling these cells with 99mTc‐hexamethylpropylenamineoxime, they were infused into the infarct‐related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% ± 3.6 and 6.8% ± 2.4 of the total infused radioactivity in the infarcted area of the heart, respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133+ and CD133−CD34+ BM‐progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium.

Pilot study to evaluate the safety and feasibility of intracoronary CD133(+) and CD133(-) CD34(+) cell therapy in patients with nonviable anterior myocardial infarction.

Objectives: The long‐term effect of intracoronary infusion of progenitor cells in patients with chronic ischemic cardiomyopathy. Background: Bone marrow stem‐cell administration in patients with myocardial infarction improved myocardial performance and in some studies contributed to favorable left ventricular remodeling. Methods: We report on the results of a pilot, single center, controlled safety, and feasibility study, including 24 patients with old, nonviable anterior myocardial infarction. Twelve patients underwent intracoronary administration of selected CD133+ and CD133−CD34+ progenitor cells and 12 were followed up on medical therapy. Left ventricular volumes and ejection fraction, at rest and during low‐dose dobutamine, and myocardial viability, using TL‐201 reinjection scintigraphy, were analyzed at baseline and long‐term follow‐up. Results: Patients in the treatment group experienced a sustained decrease in left ventricular end‐diastolic and end‐systolic resting volumes (P = 0.008 and P = 0.002, respectively), as well as an improvement in global ejection fraction at rest [from (27.2 ± 6.8)% to (29.7 ± 7.3)%, P = 0.016]. Segmental anterior and apical wall perfusion, during TL‐201 reinjection, were similarly improved (P = 0.005 and P << 0.001, respectively). One patient developed restenosis at the cell delivery site and one progression of atherosclerosis. During 28.0 ± 8.7 months of clinical follow‐up, only one patient experienced deterioration of heart failure. In the control group, we observed stability in the perfusion defect and deterioration in end‐diastolic and end‐systolic volumes (P= 0.002 and P = 0.003, respectively) and a nonsignificant decrease in ejection fraction (P = 0.11). Conclusion: Intracoronary infusion of selected CD133+ and CD133−CD34+ progenitor cells to a previously infarcted and nonviable anterior wall is safe, and results in sustained improvement in segmental myocardial perfusion and in favorable left ventricular remodeling.

Circulating levels of matrix metalloproteinase-9 (MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and their complex MMP-9/NGAL in breast cancer disease

BackgroundRecent evidence suggests that neutrophil gelatinase-associated lipocalin (NGAL) expression is induced in many types of human cancer, while detection of its complex with matrix metalloproteinase-9 (MMP-9) is correlated with cancer disease status. We aim to evaluate the serum expression of MMP-9, NGAL and their complex (MMP-9/NGAL) during the diagnostic work-up of women with breast abnormalities and investigate their correlation with disease severity.MethodsThe study included 113 women with non-palpable breast lesions undergoing vacuum-assisted breast biopsy for histological diagnosis, and 30 healthy women, which served as controls. Expression levels of MMP-9, NGAL and their complex MMP-9/NGAL were determined in peripheral blood samples with immunoenzymatic assays.ResultsWomen with invasive ductal carcinoma exhibited significantly increased levels of MMP-9, NGAL and MMP-9/NGAL compared to healthy controls (MMP-9: p < 0.003, NGAL: p < 0.008 MMP-9/NGAL: p < 0.01). Significant correlations were observed between MMP-9 and NGAL serum levels and breast disease severity score (r = 0.229, p < 0.006 and r = 0.206, p < 0.01, respectively), whereas a non-significant correlation was found for their complex. MMP-9, NGAL and their complex MMP-9/NGAL levels were not correlated with either Body Mass Index (BMI) or age of patients.ConclusionThese findings suggest that the serum measurement of MMP-9 and NGAL may be useful in non-invasively monitoring breast cancer progression, while supporting their potential role as early biomarkers of breast disease status.

Assessment of oxidative stress in patients with sickle cell disease: The glutathione system and the oxidant-antioxidant status.

Continuous reactive oxygen species (ROS) production in individuals with sickle cell disease (SCD) may alter their overall redox status and cause tissue damage. The aim of this study was to evaluate oxidative stress in patients with SCD using two new assays, FORT (free oxygen radical test) and FORD (free oxygen radical defense) along with assessment of glutathione system including superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) activities, vitamins A, C and E, malondialdehyde (MDA), non-transferrin bound iron (NTBI) and nitric oxide (NO) concentrations. A total of 40 patients with SCD and 25 apparently healthy volunteers (control group) were enrolled in the study. Components of glutathione system, vitamins A, C, and E, and malondialdehyde were determined with reverse-phase HPLC, non-transferrin bound iron (NTBI) was assessed with atomic absorption spectroscopy using graphite furnace, superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) activities were determined spectrophotometrically in red cell lysates, nitric oxide (NO) was detected colorimetrically, while FORT and FORD using colorimetric assays, as two point-of-care tests. The findings revealed significant impairment of the glutathione system indicated by reduced GSH(total) (p<0.00001), GSH(reduced) (p<0.00001) and GSSG (p>0.056) values of SCD patients compared to the control group. ROS expressed as FORT were significantly increased (p<0.00001), while antioxidant defense expressed as FORD was significantly reduced (p<0.02) in SCD group compared to the control group. Age and genotype of the patients as well as therapy of their disease appeared to play no role in their oxidative status.

Changes in free radical generation and antioxidant capacity during ultramarathon foot race

Background  Exhaustive exercise has been implicated in the generation of reactive oxygen species, resulting in oxidative stress. We studied the effect of a long‐distance, endurance exercise on oxidative stress parameters in athletes who participated in the ultramarathon race Spartathlon (246 km).

A functional hierarchy among the CD34+ hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+ CD34bright and AC133dim/-CD34+ human cord blood cells

The 5-transmembrane receptor AC133 is expressed on a subpopulation of human hematopoietic cells that includes the CD34(bright) cells. We evaluated the developmental potential of AC133+CD34(bright) and AC133(dim/-)CD34+ cells isolated from 5 cord blood (CB) samples by studying the in vitro proliferative and differentiative potential of each population in both progenitor and mature cell expansion cultures. Seven-day culture of AC133+CD34(bright) cells with a cytokine combination favoring primitive progenitor cells causes a significant increase in CD34+, CFU-C and noncycling stem/progenitor cells HPP-Q (High Proliferative Potential-Quiescent), whereas culture of AC133(dim/-)CD34+ cells shows a limited increase in committed progenitor cells only. HPP-Q cells were not found in freshly isolated AC133(dim/-)CD34+ nor in expanded CD34+ cells derived from AC133(dim/-)CD34+ cells. No statistically significant difference was observed between the 1-week expanded AC133+ and the initial AC133+CD34(bright) cells regarding their clonogenic efficiency (CE), while expanded CD34+ cells derived from AC133(dim/-)CD34+ cells exhibited a decreased CE. Subexpansion of the reselected AC133+ derived from AC133+CD34(bright) cells reveals a further increase of stem/progenitor cells and the 14-day expanded AC133+ cells reveal an unchanged CE. Subexpansion of reselected 7-day CD34+ cells derived from AC133(dim/-)CD34+ cells was not possible. Culture of AC133+CD34(bright) cells in cytokines that favor megakaryopoiesis or erythropoiesis resulted in a significant expansion of CD41+ and CD71+ cells, respectively; AC133(dim/-)CD34+, in comparison, showed a limited potential to megakaryocytic differentiation and a decreased production of erythroid cells. Our data indicate that early high proliferating stem/progenitor cells and early committed progenitors are present in AC133+CD34(bright) cells, but not in AC133(dim/-)CD34+ cells; the latter represent late committed progenitors with limited proliferative potential.

Update on the mechanism of action and on clinical efficacy of extracorporeal photopheresis in the treatment of acute and chronic graft versus host disease in children.

Extracorporeal photopheresis (ECP) has been used for treatment of steroid-refractory graft versus host disease (GVHD) with encouraging results. Although its exact mechanism of action is not fully understood, photoapheresed cells seem to induce a selective immune response directed against alloreactive T cell populations without causing generalized immunosuppression. Current pediatric experience with ECP for GVHD is available in the form of a few retrospective small studies concerning children with steroid refractory GVHD. Reviewing these data we conclude that ECP is a safe procedure, well tolerated even in low-weight pediatric patients, which warrants further evaluation in well-designed, prospective, controlled studies.

Acute resistance exercise results in catecholaminergic rather than hypothalamic-pituitary-adrenal axis stimulation during exercise in young men

Exercise is a paradigm of a stress situation. The adaptive response to stressors comprises the activation of the hypothalamic–pituitary–adrenal (HPA) axis and components of the autonomic sympathetic system. An aseptic inflammatory reaction is triggered by exercise, involving the stimulation of the so-called proinflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and IL-6. The anti-inflammatory cytokines IL-2, IL-8, and IL-10 increase moderately during resistance exercise. To investigate the effect of a short bout of resistance exercise on components of the stress and inflammatory responses during the exercise period, 17 healthy, young, untrained male volunteers were studied during 3 equal consecutive cycles of resistance exercises of 30 min total duration. Blood sampling was performed at baseline and at the end of each cycle. Lactate, cortisol, catecholamines (epinephrine, norepinephrine), IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, epidermal growth factor (EGF), and monocyte chemotactic protein-1 (MCP-1) were measured at all time-points. Circulating levels of catecholamines and lactate increased significantly (P < 0.05) whereas cortisol did not. During the time course of the exercise, circulating levels of TNFα, IL-2, and EGF increased, whereas MCP-1 decreased significantly. IL-1α, IL-1β, IL-6, IL-8, and IL-10 levels did not change significantly. Statistically significant positive linear correlations were found between areas under the curve for increases in levels of IL-2 and TNFα, TNFα and cortisol, as well as epinephrine and norepinephrine. We conclude that acute resistance exercise results in catecholaminergic, but not HPA axis stimulation during exercise, in parallel with a mild inflammatory reaction. The absence of a major inflammatory reaction and of a cortisol increase during acute resistance exercise makes this a good candidate for the exercise of sedentary individuals.

Successful Hematopoietic Stem Cell Transplantation in 2 Children with X-Linked Chronic Granulomatous Disease from Their Unaffected HLA-Identical Siblings Selected Using Preimplantation Genetic Diagnosis Combined with HLA Typing

We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor.

HLA-matched sibling stem cell transplantation in children with β-thalassemia with anti-thymocyte globulin as part of the preparative regimen: the Greek experience.

BU combined with CY, the preferred preparatory regimen for thalassemic patients, is associated with a substantial incidence of graft rejection especially in patients with advanced disease stage. This study retrospectively analyzes the outcome of 75 consecutive pediatric patients with β-thalassemia who underwent HLA-matched sibling transplantation after anti-thymocyte globulin (ATG)-containing myeloablative conditioning regimens. With a median follow-up of 9 years (range 1–15 years), the overall survival (OS) and thalassemia free survival (TFS) rates were 96% and 92%, respectively. Both the estimated TRM and the cumulative incidence of rejection/failure were 4%. The cumulative incidences of acute GVHD grade II–III and grade III were 20% and 5.3%, respectively. No patient developed acute GVHD grade IV. Only two patients developed extensive chronic GVHD. The estimated OS and TFS for patients with Class 1 and 2 disease according to Pesaro criteria were 96.3% and 94.4%, whereas for patients with Class 3 disease they were 94.1% and 88.2%, respectively. In our series, the use of myeloablative conditioning regimens, which include ATG for the transplantation of thalassemic children from matched sibling donors, resulted in excellent outcomes with very low incidences of TRM and rejection.